Agents which interact with a serotonin transporter for the treatment of cancer

ABSTRACT

The present invention relates to a method of inducing cell death in a neoplastic cell expressing a serotonin transporter (SERT) such as Burkitt&#39;s lymphoma cell or a chronic lymphocytic leukaemia cell. The method comprising exposing said neoplastic cell to a therapeutically effective amount of a pharmaceutically active agent which interacts with said SERT, said therapeutically effective amount being sufficient to cause death of said neoplastic cell.

Cancer is one of the main causes of death in the developed world. Thedisease takes many forms and affects almost all types of cell.Treatments for cancer are many and varied, and are often unpleasant andcan have serious side effects.

Burkitt's Lymphoma (BL) is a cancer endemic to the malarial belts ofequatorial Africa, North-eastern Brazil and Papua New Guinea, but whoseincidence is increasing dramatically outside these endemic regions dueto its association with HIV infection. Among North Americans there is athousand fold increase in the incidence of BL among individuals withAIDS, and this trend is likely to continue as these patients live longerthrough the provision of better treatment regimes to manage their immunedeficiency.

Burkitt's lymphoma is a highly aggressive malignancy, but is nonethelesscharacterised by a predisposition to exhibit a high degree of apoptosiswhich can be seen by its classical “starry sky” histology. This is dueto the origin of the malignancy in germinal centre (GC) cells which donot express the pro-survival protein Bcl-2 and therefore have apropensity for spontaneous apoptosis, which is carried over to the BLtumour cells.

Burkitt's lymphoma is currently treated by aggressive combinationchemotherapy, usually over a 6month period and requiring frequenthospitalisations. While there is a 3-year relapse-free survival rate ofapproximately 80% for patients with local disease, patients withdisseminated tumour respond less well to chemotherapy and have poorsurvival rates. The limited medical resources in regions where BL isendemic also limit survival. In individuals with AIDS, non-Hodgkin'slymphomas tend to be aggressive and advanced at diagnosis. The mediansurvival of HIV-positive patients with non-Hodgkin's lymphomas is 6months: Burkitt's lymphoma comprises approximately 20% of such lymphomasand is diagnosed in some 2% of AIDS patients. Intensive chemotherapy isclearly not the most desirable course of treatment for immunosuppressedindividuals.

According to a first aspect of the present invention there is provided amethod of inducing cell death in a neoplastic cell expressing aserotonin transporter (SERT), comprising exposing said neoplastic cellto a therapeutically effective amount of a pharmaceutically active agentwhich interacts with said SERT, said therapeutically effective amountbeing sufficient to cause death of said neoplastic cell.

According to a second aspect, the present invention resides in the useof an agent which interacts with a serotonin transporter expressed on aneoplastic cell in the manufacture of a medicament for the treatment ofneoplasia.

According to a third aspect, there is provided a method of treating apatient afflicted with a neoplastic condition comprising, administeringto said patient a therapeutically effective amount of a pharmaceuticallyactive agent which interacts with a serotonin transporter expressed on aneoplastic cell.

Serotonin (5-HT) is a well known neurotransmitter of the central nervoussystem which has also been implicated in diverse aspects of immuneregulation. Outside of the central nervous system (CNS) 5-HT is mainlyproduced by enterochromaffin cells of the gut and is taken up via anactive transport mechanism into a number of cell types.

It is known that the action of 5-HT, on both neuronal and other cells,can be mediated either through the many different receptor subtypes tobe found on the surface of the target cells, or via an active uptakemechanism that relies on the SERT (Mossner, R. Lesch, K. P., BrainBehav. Immun. (1998) 12: 249-271. Schorr, E. C. Arnason, G. W. BrainBehav. Immun. (1999) 13: 271-278. Barnes, N. M. Sharp, T.Neuropharmacology. (1999) 38: 1083-1152).

The SERT is expressed on cells outside of the CNS although the extent ofits distribution in the periphery is not fully established. Plateletsare by far the best-studied cell for characteristics of peripheral SERTbut other haemopoietic cells, particularly those of the immune system,also carry the SERT. These include B lymphocytes, T lymphocytes, andmacrophages. The rationale for these cells to carry functional SERT isnot known.

It has been suggested that the 5-HT uptake activity of SERT in the CNS,whilst a normal and necessary process, may be involved in neuronal celldeath, particularly in the context of pathological conditions relatingto neurodegeneration, although the mechanisms by which this occurs arenot fully understood.

The inventors have now shown that other cell types, such as BL cells aresubject to 5-HT mediated apoptosis via SERT.

The inventors have further discovered that agents which have a similarmode of action to 5-HT (e.g. d-fenfluramine) and appear to work throughSERT, can also mediate apoptosis in neoplastic cells. It will beunderstood that although the proposed mechanism of action is via SERT,the invention should not be construed as being limited thereto, and itis possible that the action of the agents may be SERT independent.

In normal individuals the amount of free circulating 5-HT is regulated,at least in part, by the SERT. In depressed individuals, the amount offree circulating 5-HT is too low. Drugs which block the uptake of 5-HTvia the SERT either selectively (known as selective serotonin re-uptakeinhibitors (SSRI's)) or non selectively help maintain an adequate levelof free circulating 5-HT and are widely used in the treatment ofclinical depression. As might be expected, administration of such drugsat doses used to prevent 5-HT uptake to neoplastic cells which wouldotherwise undergo apoptosis in the presence of 5-HT, prevents or reducessuch apoptosis.

Most surprisingly, the inventors have discovered that at higherconcentrations, i.e. at concentrations above those realised foralleviation of the symptoms of depression, 5-HT re-uptake inhibitors canactually drive apoptosis in cells expressing SERT.

Thus, it will be understood that the agent of the present invention maybe selected from 5-HT, active analogues of 5-HT, agents that have asimilar mode of action to 5-HT, selective serotonin re-uptake inhibitorsand non-selective serotonin re-uptake inhibitors.

An example of an agent which has a similar mode of action to 5-HT isd-fenfluramine.

Suitable examples of SSRI's are fluoxetine (Prozac®), paroxetine(Paxil®), citalopram (celexa®), sertraline (Zoloft®), fluvoxamine(Luvox®).

Suitable examples of non-selective re-uptake inhibitors are imipramine,desipramine, amitriptyline, chlortrytiline, clomipramine.

The methods of the present invention are particularly applicable toneoplastic cells having a relatively high susceptibility to apoptosis.Such susceptibility may be due to relatively low expression of cellsurvival genes, such as Bcl-2. Cells of this type include Burkitt'slymphoma cells and certain other lymphomas, including chroniclymphocytic leukaemia.

Alternatively, said cell may be induced to have a relatively highsusceptibility to apoptosis. Thus, it will be understood that the methodof the first aspect may include a step of increasing the susceptibilityof the neoplastic cell to apoptosis prior to, or during, exposure ofsaid cell to the pharmaceutically active agent.

Equally the method of the third aspect may include the step ofadministering to the patient an agent which increases the susceptibilityof neoplastic cells to apoptosis. Increasing susceptibility of theneoplastic cell to apoptosis may be achieved by interfering with thereplication of at least one cell survival gene, interfering with theexpression (e.g. during transcription or translation) of at least onecell survival gene and/or negating or reducing the survival function ofat least one expressed cell survival gene.

Thus, the second aspect of the present invention also resides in the useof a pharmaceutically active agent which interacts with a serotonintransporter expressed on a neoplastic cell in combination with an agentfor increasing the susceptibility of said neoplastic cell to apoptosisin the manufacture of a medicament or respective medicaments for thetreatment of neoplasia

It is known that blocking the production of the Bcl-2 gene product can,at least in vitro, lead to increased effectiveness of a number ofanticancer therapies such as chemotherapy, radiation and immunotherapy.Drugs on which the removal of Bcl-2 production has been shown to have abeneficial effect include; Paclitaxel (Taxol®), Irinotecan (Camptosar®),Fludarabine (Fludara®), Cyclophosphamide (Cytoxan®), Docetaxel(Taxotere®), Gemtuzumab ozogamicin (Mylotarg®) Cytosine arabinoside andDexamethasone. Preferably, said agent for increasing the susceptibilityof said neoplastic cell to apoptosis is an antisense drug targeted atBcl-2, an example of which is Genasense (Trademark, Genta Inc.).

In relation to said third aspect, said administration (of saidpharmaceutically active agent and/or said agent for increasing thesusceptibility of said neoplastic cells to apoptosis when present) maybe systemic or local by any convenient route e.g. orally,subcutaneously, intravenously, or by direct administration (injection orotherwise) into the neoplastic site. In general, said treatment willpreferably comprise multiple administrations whereby to maintain aneffective concentration of drug in the neoplastic cells.

Said agent (or agents) may be administered in liquid form, as a tabletor capsule, as a suspension or in solution. In each case said agent(s)may be in admixture with standard excipients, binders, taste modifiersand pH adjusters as appropriate.

It will be understood that said agent for increasing the susceptibilityof said neoplastic cell to apoptosis may be administered concomitantlywith said active agent (either combined in a single medicament or byco-administration of separate medicaments) or prior to administration ofsaid active agent. Where separate medicaments are administered(concomitantly or otherwise) the routes of administration may bedifferent.

For the treatment of anxiety or depression, SSRIs are generallyadministered orally at a dose of 20-100 mg/day, at which doses a steadystate blood level of SSRI of about 1 μM is obtained after an initialperiod. Preferably, when said agent is an SSRI, and particularly wheresaid administration is systemic, it will be administered at a level toobtain a steady state blood concentration of greater than about 1:M,more preferably greater than about 5 μM and most preferably greater thanabout 10:M. It will be understood that the skilled person will readilybe able to calculate the dosage required to achieve said steady statelevels. When said administration is non-systemic, sufficient agent willpreferably be administered to achieve a concentration in the neoplastictissue of 5 μM or more, more preferably about 10 μM or more, and mostpreferably about 25 μM or more.

It is expected that similar dosage levels will also be effective ford-fenfluramine.

Embodiments of the invention will now be described, by way of exampleonly, with reference to the accompanying diagrams in which:

FIG. 1 shows the effect of 5-HT concentration on DNA synthesis in L3055BL cells,

FIG. 2 shows the correlation between plating density and 5-HTsensitivity for L3055 cells,

FIG. 3 shows the effect on DNA synthesis in various cell lines of theaddition of 125 μM 5-HT,

FIG. 4 shows the effect of 5-HT treatment on cell cycle status in L3055cells,

FIG. 5 shows the effect of increasing dose of 5-HT on the percentage ofdead cells in a population of L3055 cells,

FIG. 6 shows the dose dependent variation in the number of viable L3055cells in a population in response to increasing 5-HT levels,

FIG. 7 shows a graphical representation of FACS analysis of L3055 cellstreated with 5-HT showing increased caspase activity,

FIG. 8 shows the inability of various 5-HT antagonists in preventing5-HT from inhibiting DNA synthesis in L3055 cells,

FIG. 9 shows the effect of low doses of SSRI's in preventing 5-HTcausing inhibition of DNA synthesis in L3055 cells,

FIG. 10 shows the inability of pargyline to prevent DNA synthesisinhibition by 5-HT,

FIG. 11 shows the same experiment as FIG. 10 replacing pargyline withclorgyline or deprenyl,

FIG. 12 shows the level of oxidatively damaged DNA in L3055 cells upontreatment with 5-HT or H₂O₂,

FIG. 13 shows western blot analysis for various BL cell lines,

FIG. 14 shows that L3055 cells carry a SERT with similar properties tothose previously described for the full length neuronal SERT.

FIGS. 15 to 17 show the dose dependent inhibitory effect of fluoxetine,paroxetine and citalopram respectively on DNA synthesis in L3055 cells,

FIG. 18 shows the effect on normal blood cells of doses of fluoxetinewhich result in tumour cell killing,

FIG. 19 shows the dose dependent inhibitory effect of imipramine on DNAsynthesis in L3055 cells,

FIG. 20 shows the effect on the survival rate of the lymphoma cellsunder increasing concentrations of fluoxetine,

FIG. 21 shows the effect of fluoxetine treatment on cell cycle status inlymphoma cells,

FIG. 22 shows the triggering of the apoptotic cascade in response totreatment with various SSRI's,

FIG. 23 shows an increase in the levels of free intracellular Ca²⁺ inresponse to treatment of L3055 cells with fluoxetine,

FIG. 24 shows the effect of SSRI's on cell killing in a BL cell linetransfected with the pro survival gene Bcl-2,

FIG. 25 shows the triggering of the apoptotic cascade in response totreatment with imipramine, and

FIG. 26 shows the effect of imipramine on cell killing in a BL cell linetransfected with the pro survival gene Bcl-2 of FIG. 24.

EXAMPLE 1 Serotonin (5-HT)

1. Action of 5-HT in Driving Apoptosis in Burkitt's Lymphoma Cells.

Monoamines, including 5-HT, have been reported to induce apoptosis incultured neuronal cells, with cerebellar granule neurons beingparticularly sensitive: indeed, serotonin-induced neuronal cell deathhas been implicated as a possible cause of neurodegenerative andneuropsychiatric disorders.

The inventors have now shown that 5-HT is a rapid promoter of programmedcell death (apoptosis) in BL lines that remain faithful to the originalbiopsy phenotype. This action of 5-HT is mediated through an activeserotonin uptake mechanism. The serotonin transporter thereforerepresents a novel therapeutic target in Burkitt's lymphoma.

1.1. Materials and Methods.

1.1.1. Cell Lines.

Group I Burkitt's lymphoma cell lines L3055 (EBV^(neg)), BL2(EBV^(neg)), Elijah (EBV^(pos)), and MUTU I (EBV^(pos)) were maintainedin early passage as described previously (Baker M P, Eliopoulos E, YoungL S, Armitage R J, Gregory C D, Gordon J. Blood (1998); 92:2830-2843).The late passage group III BL line MUTU III (EBV^(pos)) was also usedwith all cells cultured in RPMI 1640 medium supplemented with 10% SerumSupreme (BioWhittaker, Wokingham, UK), 2 mM glutamine, 100 IU/mlpenicillin and 100 μg/ml streptomycin. Stable bcl-2 and bcl-XLtransfectants of L3055 cells, together with those carrying the mammalianexpression vector pEF-MC1 neopA alone as controls, were generated andcharacterised as detailed elsewhere (Gordon J, et al. Cell Death. Diff.(2000); 7:785-794). A stable line of HEK 293 cells carrying full-lengthhuman SERT has been described previously (Qian Y, Galli A, RamamoorthyS, Risso S, DeFelice L J, Blakely R D. J. Neurosci. (1997);17:45-57).

1.1.2. Reagents.

5-HT, fluoxetine (Prozac®), pargyline, clorgyline, and deprenyl werepurchased from Sigma (Dorset, UK). The 5-HT receptor antagonistsSDZ205-557 and methysergide were obtained from Sandoz (Basel,Switzerland) and granisetron was obtained from Smith-Kline Beecham(Harlow, Essex, UK). 5-HT binoxalate, [1,2-³H(N)-] was obtained from NEN(Zaventem, Belgium). Paroxetine (Paxil®) and citalopram (Celexa®) wereobtained from Smith-Kline Beecham and Lundbeck (Copenhagen, Denmark)respectively. Mouse monoclonal anti-human SERT Ab was purchased from Mabtechnologies (Stone Mountain, USA). Affinity-purified goat F(ab′)₂antibody fragment to human IgM was purchased from ICN (Ohio, USA). JC-1(5,5′,6,6′-tetrachlorol, 1′,3,3′-tetraethylbenzimidazolyl-carbocyanineiodide) cationic dye was purchased from Molecular Probes (Leiden,Holland). The supersignal chemiluminescence reagent and horseradishperoxidase (HRP)-conjugated rabbit anti-mouse antibody were obtainedfrom Pierce (Chester, U K). Primer synthesis and DNA sequencing wereperformed by MWG Biotech (Milton Keynes, UK). Moloney murine leukaemiavirus (MMLV) reverse transcriptase and DNasel (amplification grade) wereobtained from Gibco (Paisley, UK). DNTPs and Taq polymerase wereobtained from Promega (Southampton, UK). RNAguard and oligo d(T)₁₂₋₁₈were obtained from APBiotech (Bucks., UK). RNAzol reagent was obtainedfrom Biogenesis (Poole, England). Syto 16 was obtained from MolecularProbes Europe (Leiden, The Netherlands). All other chemicals wereobtained from Sigma (Poole, UK), and were of the best grade available.

1.1.3. Assessment of DNA Synthesis.

DNA synthesis was determined by measuring ³H-thymidine ([³H]Tdr;Amersham, UK) incorporation into cellular DNA. Cells were cultured in200 μL of supplemented medium at densities and for times indicated belowin flat-bottomed, 96-well tissue culture plates (Becton Dickinson,Oxford, U K) then pulsed for the final 4 h with 50 μl of 10 μCi/ml[³H]Tdr before harvesting on a Skatron cell harvester (Helis Bio Ltd,Newmarket, UK).

1.1.4. Viability Assays.

Changes in the viability of cells cultured under conditions indicatedbelow were quantified by assessment of forward and 90° (side) lightscatter of cells as previously described (Dive C, et al. BiochimicaBiophysica Acta (1992); 1133:275-285) using an EPICS XL-MCL flowcytometer (Beckman Coulter, Miami, Fla., USA). Prior to analysis, cellswere harvested into polythene FACS tubes, and fixed at 4° C. in 400 μlof FACS buffer [PBS with 5% NGS and 2% Formaldehyde] (BDH/MERCK). Cellswere gated in two populations (viable and dead) according to theirforward and side light scatter. Viability was expressed as a percentageof the total number of cells residing in the viable and non-viablegates. Cells were also analysed according to Schuurhuis et al(Schuurhuis G J, et al. Exp. Haematol. (1997); 25:754-759) in order toassess the percentages within treated populations of those that wereviable, apoptotic, or necrotic. Syto 16 was added at a concentration of250 nM in PBS to 500,000 cells and incubated at RT for 30 min, at whichtime 5 μg/ml propidium iodide (PI) was added. Samples were then analysedimmediately by FACS and a 2D plot generated of syto 16 fluorescenceversus PI fluorescence: syto 16 is taken up only by viable cells whereasPi exclusively enters necrotic cells—syto 16^(−ve)/PI^(−ve) cells aredeemed apoptotic.

1.1.5. Cell Cycle Analysis.

Cells cultured under conditions indicated below were harvested into 100μl of 1×PBS followed by 400 μl cell cycle buffer containing Triton X-100and propidium iodide and left to incubate at 4° C. for 1 hr to allowstaining of DNA. The degree of fluorescence emitted by bound PI isdirectly proportional to the amount of DNA in each cell and was measuredby flow cytometry as previously described (MacDonald I, et al. Blood(1996); 87:1147-1154). Results are expressed as percentage of viablecells residing in each phase of the cycle.

1.1.6. Measures of Apoptosis.

Apoptosis was assessed by staining cells with acridine orange andvisualising nuclear morphology exactly as described previously (GordonJ, et al. Cell Death. Diff. (2000); 7:785-794). Viable cells display ahomogeneous chromatin-staining pattern, whereas apoptotic cellscharacteristically show condensed and fragmented chromatin.Mitochondrial depolarisation was assessed using the JC-1 cationic dye.Cells were incubated with 10 μM of JC-1 for 30 min before beingvisualised on a LSM 510 confocal microscope (Zeiss, Germany). JC-1exhibits potential-dependent accumulation in mitochondria accompanied bya shift in fluorescence emission from 525 nm (green) to 590 nm (red).Mitochondrial depolarisation is indicated by a decrease in the red/greenfluorescence ratio.

1.1.7. Detection of Caspase Activity.

Caspase activity was detected using the CaspaTag Caspase Activity Kit(Intergen, Oxford, UK). Cells cultured under the conditions indicatedbelow were harvested into 300 μl of culture medium at a cell density of10⁶cells/ml, followed by the addition of 5 μl of a 30×working solutionof FAM-VAD-FMK (a carboxy fluorescent tagged caspase inhibitor)(Intergen Co., USA). Cells were then incubated for 1 hr at 37° C. under5% CO₂ while protecting the tubes from light. At the end of theincubation, they were washed twice in 2 ml of 1×washing buffer, followedby addition of 2 μl of PI to distinguish dead cells from live cells,then analysed on an EPICS XL-MCL flow cytometer or by confocalmicroscopy. For the latter, treated cells were harvested into 300 μl ofculture medium containing 10 μl of FAM-VAD-FMK before incubating for 1hr under the conditions described above. Next, 1.5 μl of Hoechst 33342stain was added for 5 min, and cells were washed twice in 2 ml of1×washing buffer following the addition of 1 μl of PI.

1.1.8. Detection of Oxidative Damage to DNA.

Oxidative damage to DNA was quantified using the OxyDNA assay (Biotrin,Dublin, Ireland). Following culture of cells under conditions indicatedbelow, they were washed first in 1×PBS then in wash solution followed byaddition of 100 μl of blocking solution with a 1 hr incubation at 37° C.On washing twice in working solution, cells were then incubated with 100μl FITC-conjugate for 1 hr in the dark at RT before being washed twicein washing solution and once in PBS. Finally, cells were resuspended inFACS buffer and analysed on an EPICS XL-MCL flow cytometer.

1.1.9. Western Blotting.

Cell pellets were resuspended in 100 μl of lysis buffer (25 mM Tris pH7.6; 150 mM NaCl; 1 μg/ml aprotonin; 10 μg/ml leupeptin; 1 mM PMSF; 1 mMsodium orthovanadate; 1 mM EDTA and 1% Triton X-100). The samples wereincubated for 1 hr at 4° C., pelleted at high speed for 10 minutes at 4°C., and 100 μg of protein per sample were subjected to 10% SDS/PAGE.SERT-transfected HEK cells were used as a positive control. Afterrunning, gels were equilibrated in transfer buffer (25 mM Tris pH 8.3;150 mM glycine; 20% (v/v) methanol) and proteins were transferred toPVDF membranes. Blots were blocked in buffer (10 mM Tris pH 7.5; 10 mMNaCl; 0.1% Tween 20) containing 5% (w/v) milk and were probed overnightat 4° C. with a 1/5000 dilution of mouse monoclonal anti-SERT antibody.Blots were developed with an HRP-conjugated anti-mouse secondaryantibody and visualised using an enhanced chemiluminescence method.

1.1.10. 5-HT Uptake Assay.

Transport assays were performed by incubating 2×10⁷ cells with 100 nM of5-HT plus 400 nM of ³H-5-HT for 0 to 60 minutes at 37° C. in HBSScontaining 1% BSA and 10 mM HEPES (pH 7.2). The assay was stopped byfive-fold dilution in ice-cold PBS. Cell pellets were resuspended inscintillation fluid, and the total counts per pellet determined.Non-specific uptake was assayed in parallel at 4° C. under the sameconditions. The data for non-specific uptake were subtracted from thetotal uptake values to give the specific uptake.

1.2 Results.

1.2.1. 5-HT Inhibits DNA Synthesis in Group I BL Lines.

The EBV-negative L3055 line maintained in early passage has beenextensively characterised and widely used as an in vitro model forbiopsy-like BL cell behaviour. In initial experiments, L3055 cells weretreated with concentrations of 5-HT ranging from 1 to 150 μM and theirproliferative capacity assessed by [³H]-thymidine incorporation at 24,48, and 72 hours. Maximum effects were observed after 24 hr and thesedata are shown in FIG. 1. It can be seen that 5-HT caused a reduction inDNA synthesis in a concentration-dependent manner as evidenced bydecreasing [³H]-thymidine incorporation with increasing concentrationsof 5-HT.

Sensitivity to 5-HT was found to correlate with the number of cellsplated: thus, whereas >50 μM 5-HT was required to achieve 50% inhibitionof DNA synthesis when cells were cultured at the relatively high platingdensity of 10⁵ per ml (2×10⁴/well), when plated at 2.5×10⁴ per ml (5×10³per well), 50% inhibition was achieved with around 5 μM 5-HT. Likewise,FIG. 2 shows that at a fixed 5-HT concentration of 50 μM (indicated byarrow A), an approximate 35%, 70%, and >90% inhibition of DNA synthesiswas achieved for cells plated at 2×10⁴, 10⁴, and 5×10³ per wellrespectively. As would be expected, lower concentrations resulted inless inhibition (arrow C, 5 μM) and higher concentrations resulted ingreater inhibition (arrow B, 150 μM). Particularly in very earlypassage, even 1 μM 5-HT could effect a significant reduction in thelevel of DNA synthesis otherwise occurring in L3055 cells at lowernumbers (data not shown). This suggests that relatively low doses couldbe more effective if the tumour burden can be reduced, for example incombination with alternative therapies.

Three other group I BL cell lines were also investigated, namely: MutuI, Elijah, and BL2. FIG. 3 shows that when these were treated with ahigh concentration (125 μM) of 5-HT for 24 hr of culture, a substantialdecrease in DNA synthesis as measured by [³H]-thymidine incorporationwas again noted. In contrast, Mutu III, an apoptosis resistant (groupIII) BL cell line, showed only marginally decreased DNA synthesis inresponse to 5-HT. The unrelated T-cell leukaemia line, Jurkat, was foundto be insensitive to 5-HT actions (not shown).

It is well documented that cessation of DNA synthesis can be achieved ingroup I BL cells through the engagement of BCR (B cell receptors) whichis accompanied by growth arrest in the G₀/G₁ compartment of the cellcycle and subsequent entry into programmed death. The inventors haveshown that 5-HT similarly encouraged arrest at a distinct phase of thecell cycle. FIG. 4 compares the cell cycle status of L3055 cells after24 hr treatment with 5-HT or anti-μ chain antibody an antibody to BCR(to ligate the BCR) (columns A—control, columns B—5-HT 150 μM, columnsC—anti-μ chain antibody 10 μg/ml): each was used at its maximalconcentration for inhibiting DNA synthesis and inducing cell death.Although BCR-ligation exerted a somewhat greater influence on the cellcycle profile, both treatments resulted in an accumulation of arrestingcells in the G₀/G₁ compartment with a corresponding disappearance fromboth the S and G₂/M phases of cycle compared to control cultures.

1.2.2. 5-HT-promoted Inhibition of DNA Synthesis in Group I BL Lines isAccompanied by Cell Death.

In order to ascertain whether 5-HT-promoted cessation of DNA synthesiswas accompanied by cell death, L3055 cells were again treated with 1 to150 μM of 5-HT, cultured for 24 hr but then at the end of theincubations analysed for forward versus side scatter profile by FACS.The cells were gated into two populations based on different lightscatter properties: large live cells and shrunken dead cells. As seenfrom FIG. 5, 5-HT treatment resulted in a dramaticconcentration-dependent increase in the number of L3055 cells acquiringlight scatter characteristics of dead cells (indicated by arrow A) overlive cells (indicated by arrow BY, the percentages in the Figurerepresenting the percentage of live cells. The background presence of20-25% dead cells is typical of early passage BL lines maintainingbiopsy traits (see control panel). In an analogous set of experiments(on somewhat earlier passage cells), 5-HT treated cells were doublestained with syto 16 and propidium iodide (PI): syto 16 is taken up onlyby viable cells whereas PI only enters cells whose membranes have becomepermeabilised. It can be seen from FIG. 6 that the number of viablecells (indicated by arrow A) (syto 16^(+ve)/PI^(−ve)) decreased in aconcentration-dependent manner in response to serotonin with a trendsimilar to that assessed from changes in light scatter propertiesdescribed above. By the end of a 24 hr exposure to 5-HT, approximatelyequal numbers of cells had assumed characteristics of necrosis(indicated by arrow B) (syto 16^(−ve)/PI^(+ve)) versus apoptosis(indicated by arrow C) (syto 16^(−ve)/PI^(−ve)).

1.2.3. Characteristics of 5-HT-promoted Apoptosis in Group I BL Lines.

The staining of the 5-HT-treated L3055 cells with the carbocyanineliquid crystal forming probe JC-1 highlighted a loss of mitochondrialmembrane potential accompanying the 5-HT-induced apoptosis of group I BLcells. Thus, following exposure to 5-HT, there is a completedisappearance of the intense red emission from JC-1 aggregates which incontrol cells form within mitochondria maintaining their membranepotential integrity. Instead, 5-HT-treated cells develop a diffuse greenemission that results from the disseminated distribution of relativelyunconcentrated JC-1 monomers (not shown).

Another hallmark of programmed cell death is caspase activationalthough, for some B lymphocyte subsets, caspase-independent routes toapoptosis have been described. To assess whether the caspase cascade wastriggered by 5-HT in L3055 cells, the carboxyfluorescent probeFAM-VAD-FMK was used. This probe binds irreversibly to caspases in theiractive configuration. Increased activation of caspases over backgroundlevels in L3055 group I BL cells is demonstrated by 6 hrs of treatmentwith 5-HT. FIG. 7 illustrates this increase by FACS-based analysis.Panel A shows the relative percentage of cells staining positive (peakII) and negative (peak I) for caspase activity prior to treatment with5-HT. Panel B shows the same data after 6 hr treatment with 5-HT. TheFigure show an increase in the percentage of cells showing caspaseactivity from a background level of 29% to 59% after 6 hr 5-HTtreatment.

1.2.4. Selective Serotonin Re-uptake Inhibitors Inhibit Actions of 5-HTon Group I BL Cells at Relatively Low Levels.

The actions of 5-HT, on both neuronal cells and lymphocytes, can bemediated either through the many different receptor subtypes to be foundon the surface of the target cells or via an active uptake mechanismthat relies on the serotonin transporter (SERT). To explore which ofthese pathways was responsible for the observed effects of 5-HT on groupI BL lines, L3055 cells were pre-treated with a range of 5-HT receptorantagonists and selective serotonin re-uptake inhibitors (SSRI's). Threereceptor antagonists were tested: SDZ205-557, a 5-HT₄ receptorantagonist; granisetron, a highly selective 5-HT₃ receptor antagonist;methysergide which antagonises 5-HT_(1A), 5-HT_(1B), 5-HT_(1D),5-ht_(1E), 5-ht_(1F), 5-HT_(2A), 5-HT_(2B), 5-HT_(2C), 5-ht_(5A),5-ht_(5B), 5-HT₆ and 5-HT₇ receptors. FIG. 8 shows that all threereceptor antagonists (column A—control (no receptor antagonist), columnB—SDZ, column C—methysergide, column D—granisetron, column E—combined),at concentrations considerably higher than their affinities for therespective 5-HT receptor subtypes, and even when combined, failed toinfluence the ability of 5-HT to inhibit DNA synthesis in L3055 cells(compare control (no 5-HT) columns with 5-HT columns). By contrast, FIG.9 shows that three structurally distinct SSRI's, fluoxetine (columns A),citalopram (columns B), and paroxetine (columns C) each substantiallyreversed, in a concentration-dependent manner, the 5-HT-dependentcessation of DNA synthesis that was otherwise provoked (column I). Therank order and relative potencies for each of the SSRI inhibiting5-HT-promoted cessation of DNA synthesis in L3055 cells correlated wellwith their known IC₅₀ values for blocking 5-HT uptake by neuronal SERT:fluoxetine=6.8 nM; citalopram=1.8 nM; paroxetine=0.29 nM.

The actions of the SSRI's on 5-HT-dependent inhibition of DNA synthesiswere reflected in their ability to reverse both 5-HT-dependent celldeath and caspase activation in L3055 cells. As seen from Table 1,optimal concentrations of each of the three SSRI's were able to blocksubstantially the loss of viability arising from a 24 hr exposure to5-HT as judged by changes in forward versus side scatter properties ofthe cells. Similarly, 5-HT-induced caspase activation at 6 hr was almostcompletely abolished by pre-treatment of cells with each of the SSRI'sstudied. TABLE 1 Reversal of 5-HT actions by SSRI % viable cells^(b) %caspase activation^(c) Treatments^(a) (24 h) (6 h) Control 69.3 ± 1.2027.3 ± 1.20 5-HT 21.3 ± 8.65 58.3 ± 2.03 Fluoxetine + 5-HT 54.7 ± 0.8830.3 ± 2.91 Paroxetine + 5-HT 58.0 ± 3.06 29.3 ± 4.98 Citalopram + 5-HT55.3 ± 4.98 29.3 ± 1.33^(a)L3055 cells were cultured with or without 5-HT (150 μM) as indicatedand SSRI: fluoxetine (5 μM); paroxetine (2 μM); citalopram (10 μM).^(b)At the end of a 24 h culture period with treatments shown, cellswere analysed by FACS for forward versus side light scatter propertiesas in FIG. 5. The % viable cells remaining are given as means ± S.E.M.of 3 experiments.^(c)After 6 hr of treatment, L3055 cells were stained with FAM-VAD-FMKand analysed for active caspases as for FIG. 7. Results are given as %cells active caspase positive and are represented as means ± S.E.M. of 3experiments.1.2.5. Evidence that 5-HT-induced apoptosis is not due to oxidativestress

When 5-HT is transported into cells it can either be stored in vesiclesuntil released, or converted to 5-hydroxyindoleacetic acid (5-HIAA) bythe mitochondrial enzyme monoamine oxidase (MAO). This enzyme exists intwo forms, MAO-A and MAO-B. As 5-HT catabolites are highly oxidative,the inventors have assessed whether, as described for some neuronalcells, 5-HT is signalling group I BL cells to undergo apoptosis byinducing oxidative stress. First, the effect of pre-treating cells withvarious concentrations (FIG. 10: column A—0, column B—10 μM, column C—25μM, column D—50 μM, column E—100 μM) of the relatively broad-acting MAOinhibitor pargyline prior to adding 5-HT was investigated. As shown inFIG. 10, even the highest concentration of 100 μM pargyline (column E),which had no deleterious effect by itself (“control”), failed toinfluence 5-HT-induced cessation of DNA synthesis (“+5-HT”). FIG. 11shows that the same outcome was observed when using clorgyline anddeprenyl which, respectively, display high selectivity inhibition forthe MAO-A and MAO-B isoforms (column A—0, column B—clorgyline 10 μM,column C—clorgyline 1 μM, column D—deprenyl 10 μM, column E—deprenyl 1μM, column F—clorgyline/deprenyl 10 μ, column G—clorgyline/deprenyl 1μM.

While the above experiments offer evidence that oxidative stress was notthe cause of 5-HT-induced apoptosis in BL cells, further more directexamination of the generation of potential reactive oxygen species (ROS)as a consequence of 5-HT uptake was undertaken. For this the OxyuDNAassay was used, which quantifies 8-oxo-7,8-dihydro-2′-deoxyguanosine(8-oxodGuo) residues that are generated as a result of oxidative damageto DNA (see methods section 1.1.8 above). Referring to FIG. 12, it canbe seen that there was no increase in the level of oxidatively damagedDNA on treating L3055 cells with concentrations of 5-HT that otherwiseeffectively promoted apoptosis (panel A, bold plot represents cellscultured in control medium for 6 hr, faint plot represents cellscultured in medium containing 150 μM 5-HT for 6 hr. Reference to panel Areveals that there was no increase in the number of cells conjugating tothe fluorescent antibody). By contrast, treating cells with 30 μM H₂O₂,which also efficiently promoted apoptosis (data not shown) resulted in alarge increase in the level of 8-oxoguanosine residues generated (panelB, bold plot as for Panel A, faint plot represents cells cultured inmedium containing 30 μM H₂O₂. Panel B shows an increase in the number ofcells fluorescing, therefore indicating a greater proportion of cellscontaining oxidative damage).

1.2.6. Burkitt's Lymphoma Cells Express a Functional SerotoninTransporter.

The reversal of 5-HT action on group I BL cells by SSRI's indicates that5-HT is exerting its effects via an active transport mechanism. Althoughthe presence of SERT has been described for both EBV-transformed Blymphoblasts and normal peripheral blood lymphocytes, there have been noprevious reports of its existence or characteristics on lymphoma cellsof any description.

FIG. 13 shows western blot analysis of cell lysates with an antibodyraised to a unique sequence within SERT. This revealed the presence ofprotein migrating as a single band with an approximate apparentmolecular weight of 70 kDa in all BL lines used in this study. This bandran faster than immunoblotted protein from HEK293 cells that had beentransfected with human neuronal SERT; wild-type HEK293 cells werenegative for immunoreactive protein. Alternative post-translationalmodification of SERT in different cell types has been suggested toaccount for the varying size range reported for the transporter,including that of a prominent 60 kDa species in some tissues.Importantly, the Mutu III line which was relatively resistant to theinhibitory actions of 5-HT (see FIG. 2) nevertheless carried equivalentlevels of SERT protein to its group I counterpart, and fully resistantL3055 bcl-2-transfectants also retained high levels of SERT. FIG. 13also shows that while the group I Elijah BL cells consistently displayeda lower level of SERT protein than the other BL lines, a demonstrableimmunoreactive band was always present. When assessed for first orderkinetics of 5-HT uptake as shown by FIG. 14, L3055 cells were shown tocarry a transporter with properties similar to those previouslydescribed for full-length neuronal SERT.

EXAMPLES 2 TO 4

2. Action of SSRI's in Driving Apoptosis in Burkitt's Lymphoma Cells.

2.1 Results.

2.1.1. SSRI's Inhibit DNA Synthesis and Induce Cell Death in Group IBurkitt's Lymphoma (L3055) Cell Lines.

FIGS. 15 to 17 show the dose dependent inhibitory effect of fluoxetine(Example 2), paroxetine (Example 3) and citalopram (Example 4)respectively on DNA synthesis in the group I BL cell line (L3055). Thestudies used a single dose of drug (dose indicated on a log₁₀ scale) andassessed inhibition of DNA synthesis, a prelude to apoptosis, by[³H]-thymidine incorporation at 24 hrs.

The concentrations of SSRI's needed to effect killing of lymphoma cellsin vitro were in the order of 10- to 20-fold higher than those achievedcurrently in the circulation of patients being administered the drugsfor anti-depressant therapy.

Patients on SSRI's for depression sustain their circulating levels formonths on repeated administrations. Also, circulating levels arebelieved to represent a gross underestimation of the accumulation ofSSRI in body tissues. The lymphoma are, by definition, tumours oflymphoid tissues (e.g. lymph nodes). FIG. 18 shows the effect of variousdoses of fluoxetine on normal peripheral blood mononuclear cells (PBMC)after culture for 24 and 48 hours in media containing the drug (columnA—0, column B—30 μM, column C—20 μM, column D—10μM, column E—5μM columnF—1 FM, column G—0.5 μM). Importantly, this shows that normal bloodcells are immune to the effects of fluoxetine at concentrations where itkills the tumour cells.

FIG. 20 shows an identical experiment to that shown in FIG. 6, replacingculture in media containing 5-HT with culture in media containingfluoxetine. The Figure shows that increasing the dose of the SSRIfluoxetine which was administered to populations of L3055 cells directlydrove the lymphoma cells to die and again, that after 24 hrs similarnumbers of cells had entered apoptosis and necrosis (quadrant A livecells, quadrant B cells entering necrosis, quadrant C cells enteringapoptosis). After 24 hours a dose of 10-15 μM fluoxetine resulted inabout 40% of the lymphoma cells entering apoptosis.

2.1.2. Changes in Cell Cycle Profile in Response to Addition of SSRI's.

FIG. 21 compares the cell cycle status of L3055 cells after 24 hrtreatment with the SSRI fluoxetine or anti-μ chain antibody (to ligatethe BCR) (column A—control, column B—fluoxetine 10 μM, column C—anti μchain antibody 10 μg/ml): each was used at its maximal concentration forinhibiting DNA synthesis and inducing cell death. As with the sameexperiment performed using 5-HT, BCR-ligation exerted a somewhat greaterinfluence on the cell cycle profile, but both treatments resulted in anaccumulation of arresting cells in the G₀/G₁ compartment with acorresponding disappearance from both the S and G₂/M phases of the cellcycle compared to control cultures. These changes are comparable withthose associated with a cessation of DNA synthesis and entry intoapoptosis.

2.1.3. Action of SSRI's in Inducing Caspase Activity and DisruptingMitochondrial Membrane Potential.

FIG. 22 shows by FACS-based analysis using FAM-VAD-FMK staining thattreatment of cells with SSRI's (single dose for 24 hours) triggerscaspase activation (peak I number of cells staining negative for caspaseactivity, peak II number of cells staining positive for caspaseactivity) one of the key components of the apoptotic cascade with panelA being a control, panel B—fluoxetine treatment (10 μM), panelC—paroxetine (10 μM), and panel D—citalopram (100 μM). From FIG. 22, itcan be seen that all of the drugs have a significant effect on thenumbers of cells showing caspase activity.

A major down-stream substrate of the caspase pathway, Poly(ADP-ribose)polymerase-1 (PARP-1), undergoes cleavage in biopsylike BL cells as aresult of fluoxetine action. Whereas intact PARP-1 was the almostexclusive form in control cells, 20 μM fluoxetine promoted extensivePARP-1 cleavage.

Annexin V binding to phosphatidylserine exposed on the outer leaflet ofcells to signal for phagocytic engulfment is another commonly usedmeasure of programmed death although caution should be applied whenusing as a sole indicator for assessing apoptosis in B cells. It wasfound that while untreated L3055 cells displayed low level Annexin Vbinding on approximately half the cells, the majority of cells developeda higher level of binding following their culture with fluoxetine (Table2). TABLE 2 Influence of fluoxetine on phosphatidylserine exposure andDNA strand breaks in L3055 BL cells Annexin V binding^(b) Treatment^(a)% positive MFI % TUNEL positive^(c) Control 52.3 ± 1.8  3.9 ± 0.2 17.8 ±3.7 Fluoxetine 81.1 ± 3.9 26.1 ± 8.4 35.7 ± 7.6^(a)L3055 cells were cultured at 10⁶/ml for 17 h in control medium, andwith Fluoxetine at 20 μM.^(b)Samples analysed by FACS to generate % positive cells within theAnnexin V^(+ve)/PI^(−ve) “apoptotic” gate together with Mean FluorescentIntensity (MFI) of stain. Results are Means ± SE of 3 separateexperiments,^(c)Samples analysed by FACS to generate % non-subdipolid cells (asassessed by PI stain) positive by TUNEL (terminal deoxynucleotidyltransferase (TdT)-mediated dUTP-biotin nick end-labelling). Results areMeans ± SE of 3 separate experiments.

Neither BL cells nor their normal germinal centre B cell equivalentstended to produce the extensive “DNA ladders” that can be seen as aresult of DNA fragmentation when other cells (e.g. thymocytes) undergoapoptosis. Nevertheless, whether fluoxetine might be inducing the typeof DNA breaks associated with apoptosis was ascertained by using aTUNEL-based method where cells were first treated with DNA ligase beforeassessing the incorporation by TdT of a fluorescent-labelled nucleotideinto DNA with strand breaks having blunt ends or single base overhangs.The results showed a discernible increase in the number of L3055 cellswith such breaks following their treatment with fluoxetine (Table 2).

Importantly, normal peripheral blood mononuclear cells (PBMC) remainedviable on exposure to SSRI. The viability of cells exposed for 24 h to 5μM fluoxetine and to 10 μM fluoxetine was recorded as 97.3±0.88% and97.3±0.33% respectively compared to 98.0±0.58% in control cultures. Evenwith 20 μM fluoxetine, viability remained at 83.7±1.20%. A nearidentical outcome was noted with normal resting B cells isolated fromtonsils where viabilities after 24 h of culture with control medium orfluoxetine at 5, 10, or 20 μM were 97.4±0.53%, 97.0+0.41%, 96.5±0.88%and 85.9±1.07% respectively. To assess the possibility that SSRI mightselectively target B cells when actively cycling, tonsilar B cells wereexposed overnight to fluoxetine following 2 days of stimulation eitherwith Staphylococcus aureus Cowan (SAC) (alone or combined with solubleCD40L) or with phorbol 12-myristate 13 acetate (PMA) (alone or incombination with ionomycin). Little inhibition of DNA synthesis occurredin any of the actively cycling populations even with fluoxetine presentat 20 μM. Similarly, the clear inhibitory effect of fluoxetine on DNAsynthesis in biopsylike BL lines such as L3055 was not evident whenassessed on 5 non-BL lines with either no, or only minor reductions, inthe level of ³H-Thymidine incorporation observed in Nalm-6 (“pre-B”),RPMI 8226 (“plasmacytoid”), or the three T-cell lines: Jurkat, J10, andCEM (data not shown).

2.1.4. Effect of SSRI's on Intracellular Free Ca²⁺.

FIG. 23 shows that treatment of L3055 cells with fluoxetine (indicatedon a log₁₀ μM scale) leads to a sustained increase in the levels ofintracellular free Ca²⁺. Fluoxetine, paroxetine and citalopram also allpromoted-an increase in basal Ca²⁺ with a similar shape and kinetics(data not shown). Further studies with fluoxetine have indicated that:(i) it acts on thapsigargin-sensitive endoplasmic reticulum Ca²⁺ storesand; (ii) triggers Ca²⁺ influx. The increase in intracellular free Ca²⁺is known to be an important step in the induction of apoptosis. Thisfurther supports data suggesting that drugs which target SERT can induceapoptosis in malignant cells which do not express tumour survival genes.

2.1.5. Effect of the Tumour Survival Gene Bcl-2 on the Apoptotic Effectof SERT Interacting Drugs.

FIG. 24 shows forward versus side scatter FACS profiles showing theeffect on the survival of transfecting native BL cell lines, which aresusceptible to killing by the SSRI's (SSRI concentrations are the sameas those used in FIG. 22, cells were exposed to the drugs for 24 hrs).In FIG. 24, arrows A indicate dead cells, arrows B indicate live cells,with the percentages given relating to the percentage of live cells.Panel I shows data for the native BL cell line and panel II for theBcl-2 transfected cell line. As can be seen, cell killing is greatlyreduced in the presence of the Bcl-2 gene product. Since this gene isexpressed in most peripheral cells they will be relatively resistant toSERT interacting drug induced apoptosis.

EXAMPLE 5 Imipramine

3. Action of the non-Selective Re-uptake Inhibitor Imipramine in DrivingApoptosis in BL Cells

FIG. 19 shows the dose dependant effect of imipramine on DNA synthesisin group I BL cell lines (single dose, DNA synthesis measured after 24hrs). Imipramine although not an SSRI is known to interact with SERTwith a similar affinity as fluoxetine. This can be seen by the similarEC₅₀ values for DNA synthesis of the two drugs.

FIG. 25 corresponds to FIG. 22, and shows that imipramine also has asignificant effect on the number of cells exhibiting caspase activity(peak I cells staining negative, peak II cells staining positive).

Referring to FIG. 26, it can be seen that, as was the case for theSSRI's (FIG. 24), cell killing by imipramine is greatly reduced for theBcl-2 transfected BL cell line (panel II: 91% survival) over the nativecell line (panel I: 30% survival).

4. Possible Role of c-myc

BL is characterised by translocations of one c-myc allele to one of theimmunoglobulin loci and the extraordinarily high growth rate thatcharacterises these tumours reflects the pro-proliferative actions ofthe translocated gene. Despite its hallmark translocation toimmunoglobulin loci, the BL-associated constitutive expression of c-mycremains dependent on the binding of the recognised c-myc transcriptionfactor Nm23-H2 to a PuF site within the regulatory sequence of thetranslocated gene. Using real-time RT-PCR (data not shown), it was foundthat a 6 h exposure of biopsylike BL cells to fluoxetine resulted in anapproximate 60% reduction in the levels of both nm23-H2 and c-myc. Itwas also observed that there was a rapid 75% decrease in the expressionof nm23-H1 and mRNA, the product of which has also been linked to c-mycexpression. In contrast expression of the house-keeping gene cyclophilinA was not significantly altered with levels remaining at 81.8% (SE=8.3%)of control values following fluoxetine treatment (P=0.1 6).

Recently Nm23-H1 has been identified as possibly being linked to c-mycexpression. Thus the SSRI appear to influence the very genes thatunderpin the uncontrolled cell division normally associated with BL.Furthermore, high expression of Nm23-H1 has been correlated with poorresponses to treatment in high-grade lymphomas other than BL. Theseobservations may therefore have implications for the clinicalexploitation of SSRI in the broader context of B cell lymphoma.

1. A method of inducing cell death in a neoplastic cell expressing aserotonin transporter, comprising exposing said neoplastic cell to atherapeutically effective amount of a pharmaceutically active agentwhich interacts with said SERT, said therapeutically effective amountbeing sufficient to cause death of said neoplastic cell.
 2. The methodaccording to claim 1, wherein the pharmaceutically active agent isselected from 5-HT, an active analogue of 5-HT, an agent that has asimilar mode of action to 5-HT, a selective serotonin re-uptakeinhibitor and a non-selective serotonin re-uptake inhibitor.
 3. Themethod according to claim 2, wherein said SSRI is selected from,fluoxetine, paroxetine, citalopram, sertraline, and fluvoxamine.
 4. Themethod according to claim 2, wherein said non-selective re-uptakeinhibitor is selected from, imipramine, desipramine, amitriptyline,chlortrytiline, and clomipramine.
 5. The method according to claim 2,wherein said agent which has a similar mode of action to 5-HT isd-fenfluramine.
 6. The method according to any claim 1, which comprisesthe further step of increasing the susceptibility of the neoplastic cellto apoptosis prior to, or during, exposure of said cell to thepharmaceutically active agent.
 7. The method according to claim 6,wherein the increase in susceptibility of said neoplastic cell toapoptosis is achieved by contacting the cell with an agent which reducesBcl-2 expression.
 8. The method according to claim 7, wherein said agentis an antisense agent targeted at Bcl-2.
 9. The method according toclaim 1, wherein the neoplastic cell is a Burkitt's lymphoma cell or achronic lymphocytic leukaemia cell. 10-18. (canceled)
 19. A method oftreating a patient afflicted with a neoplastic condition comprising,administering to said patient a therapeutically effective amount of apharmaceutically active agent which interacts with a serotonintransporter expressed on a neoplastic cell.
 20. The method of treating apatient according to claim 19, wherein the pharmaceutically active agentis selected from 5-HT, an active analogue of 5-HT, an agent that has asimilar mode of action to 5-HT, a selective serotonin re-uptakeinhibitor, and a non-selective serotonin re-uptake inhibitor.
 21. Themethod of treating a patient according to claim 20, wherein said SSRI isselected from the group consisting of, fluoxetine, paroxetine,citalopram, sertraline, and fluvoxamine.
 22. The method of treating apatient according to claim 20, wherein said non-selective re-uptakeinhibitor is selected from the group consisting of, imipramine,desipramine, amitriptyline, chlortrytiline, and clomipramine.
 23. Themethod of treating a patient according to claim 20, wherein said agentwhich has a similar mode of action to 5-HT is d-fenfluramine.
 24. Themethod of treating a patient according to claim 19, wherein the methodfurther comprises the step of administering to said patient an agentwhich increases the susceptibility of neoplastic cells to apoptosis saidadministration being prior to, or during, exposure of said patient tothe pharmaceutically active agent.
 25. The method of treating a patientaccording to claim 24, wherein said agent for increasing thesusceptibility of said neoplastic cells to apoptosis is an agent whichreduces Bc1-2 expression.
 26. The method as claimed in claim 25, whereinsaid agent is an antisense agent targeted at Bcl-2.
 27. The method oftreating a patient according to claim 20 wherein, said pharmaceuticallyactive agent is an SSRI.
 28. The method of treating a patient accordingto claim 27 wherein, said SSRI is administered systemically at a levelto obtain a steady state blood concentration of greater than 1 μM andpreferably greater than 5 μM.
 29. The method of treating a patientaccording to claim 27 wherein, said SSRI is administerednon-systemically, so as to achieve a concentration in the neoplastictissue of 5 μM or more, preferably 10 μM or more.
 30. The method oftreating a patient according to claim 20 wherein said pharmaceuticallyactive agent is d-fenfluramine.
 31. The method of treating a patientaccording to claim 30 wherein, d-fenfluramine is administeredsystemically, at a level to obtain a steady state blood concentration ofgreater than 1 μM and preferably greater than 5 μM.
 32. The method oftreating a patient according to claim 30 wherein, d-fenfluramine isadministered non-systemically so as to achieve a concentration in theneoplastic tissue of 5 μM or more, preferably 10 μM or more.
 33. Themethod of treating a patient according to claim 19, wherein theneoplastic cell is a Burkitt's lymphoma cell or a chronic lymphocyticleukaemia cell.